Qing Fei Hua Xian Decoction ameliorates bleomycin-induced pulmonary fibrosis by suppressing oxidative stress through balancing ACE-AngII-AT1R/ACE2-Ang-(1-7)-Mas axis

Objective(s): We aimed to investigate the preventative effect of Qing Fei Hua Xian Decoction (QFHXD) against pulmonary fibrosis (PF) and its potential mechanisms. Materials and Methods: Bleomycin (BLM)-induced rats were respectively treated with 413.3, 826.6, and 1239.9 mg/kg of QFHXD and prednisone for 28 days. The lung tissues of rats were collected on day 28 for histological and western blotting analysis. Results: QFHXD significantly reduced alveolus inflammation, collagen accumulation, and fibrosis deposition in BLM-induced PF rats (P<0.05). Collagen I and III, vimentin, and α-smooth muscle actin(α-SMA) expression levels were likewise decreased in PF rats treated with QFHXD (P<0.05). Additionally, QFHXD increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) while decreasing NADPH oxidase 4 (NOX4) expression (P<0.05). Furthermore, QFHXD suppressed the PF progression by down-regulating Angiotensin-Converting Enzyme (ACE) -Angiotensin II (AngII) -Angiotensin II Type 1 Receptor (AT1R) axis (P<0.01) and up-regulating Angiotensin-Converting Enzyme 2 (ACE2) -Angiotensin-(1-7) (Ang-(1-7)) -Mas axis (P<0.05). Conclusion: QFHXD suppressed inflammatory infiltration and PF brought on by BLM in lung tissues through reducing oxidative stress by maintaining the equilibrium of ACE-AngII-AT1R and ACE2-Ang-(1-7) -Mas axes. This study may provide a novel clinical therapy option for PF.


Introduction
Pulmonary fibrosis (PF) is a progressive, fatal, and chronic disorder marked by inflammatory infiltration of the lungs and fibrosis of the lung parenchyma (1). Patients with PF gradually lose lung function as the disease worsens, which may lead to respiratory failure or even death (2). To date, only pirfenidone and nintedanib, and lung transplantation have been proven to be effective treatments for PF globally (3), and no drugs have shown a predicted survival advantage for PF. Therefore, novel medicines are urgently required to slow the progression of PF and improve the quality of life for PF patients.
Oxidative stress is associated with reactive oxygen species (ROS) and reactive nitrogen species (RNS) overproduction, leading to oxidation/anti-oxidation disequilibrium (4). Idiopathic pulmonary fibrosis (IPF) progresses due to an excess of ROS and activation of multiple NADPH oxidase (NOX) isoforms (5). Elevated levels of ROS induce PF through pathological processes, such as alveolar epithelial cell (AEC) apoptosis, inflammatory cell infiltration, collagen accumulation, and epithelial-mesenchymal transition (EMT) (6). NADPH oxidase 4 (NOX4), one of the NOX family oxidoreductases, is crucial for PF development by inducing intracellular ROS generation, AEC death, Smad phosphorylation, and extracellular matrix (ECM) production (7,8). Numb expression is suppressed by nuclear factor erythroid 2-related factor 2 (Nrf2) to affect EMTmediated PF via anti-oxidant pathway (9), and deficits of Nrf2 are associated with the onset of PF. Moreover, NOX4-Nrf2 imbalance has been discovered in the lung tissue of PF patients, and this suggests that a treatment approach aimed at restoring the NOX4-Nrf2 redox equilibrium in PF might be effective (10).
Qing Fei Hua Xian Decoction (QFHXD) was constructed to treat PF based on Chinese medical theory and clinical experience related to lung diseases. QFHXD is composed of 14 herbs, namely Astragalus membranaceus, Angelica sinensis, Ephedra, Amygdalin, Pinellia, Whole trichosanthes kirilowii, Radix pseudostellariae, Semen lepidii, Radix paeoniae rubra, and Thunberg fritillary bulb, etc. A number of these herbal drugs have been well-documented to exhibit significant anti-fibrosis and anti-oxidant properties. For instance, Angelica sinensis polysaccharide has been found to inhibit the EMT progression of IPF by downregulating the expression of differentiation antagonizing non-protein coding RNA (DANCR) and suppressing AUbinding factor 1 (AUF1)-mediated FOXO3 translation (14). Amygdalin inhibited the transforming growth factor-β1 (TGF-β1) expression and suppressed small mothers against decapentaplegic (Smad)2/3 phosphorylation to slow down the EMT process (15). Heterophylline B extracted from Radix pseudostellariae inhibited BLM-induced PF, possibly by down-regulating TGF-Smad2/3 and adenosine 5-monophosphate-activated protein kinase (AMPK)mediated stimulator of interferon genes (STING) signaling pathways (16). Total paeony glucosides isolated from the roots of Radix paeoniae rubra have been shown to protect the anti-oxidant defense system against oxidative stressinduced diseases (17). However, the underlying mechanisms of QFHXD against PF remain unclear.
In this study, we hypothesized that QFHXD could play a protective role in PF by attenuating oxidative stress, possibly by modulating ACE/AngII/AT1R and ACE2/Ang-(1-7)/Mas axes, given the link between PF pathogenesis and oxidative stress and the ACE-AngII-AT1R/ACE2-Ang1-7-Mas axis. Here, BLM-induced PF rat models were used to assess the preventative effects of QFHXD against oxidative stress and PF. Moreover, the influence of QFHXD on BLMinduced PF development in vivo and the anti-oxidative actions of QFHXD through restoring ACE-AngII-AT1R/ ACE2-Ang1-7-Mas axis homeostasis were clarified. The findings in this study would provide theoretical support for the clinical treatment of PF.

Herbal medicines and reagents
Jingpai Chizheng Tang Pharmaceutical Co. LTD.

Animals
Two-month SD male rats (200-220 g) were bought from Hunan SJA Laboratory Animal Co., LTD. (

BLM-induced PF rat models and drug administration
Using a random number generator, 60 male SD rats were assigned to six groups (n = 10 in each): control, fibrosis, prednisone, QFHXD-413.3 mg/kg, QFHXD-826.6 mg/kg, and QFHXD-1239.9 mg/kg. In the fibrosis, prednisone, and QFHXD groups, PF rats were generated by intraperitoneally injecting 1.5% isoflurane anesthesia and then receiving 5 mg/kg of BLM with sterile 0.9% saline endotracheally. The control rats received the same amount of sterile saline. For Animal Equivalent Dose (AED, mg/kg), we multiplied the human dosage (mg/kg) by the K m ratio. QFHXD is administered in doses of 4,000 mg/60 kg to adults according to the drug instruction, while prednisone is in doses of 10 mg/60 kg to adults. On day 1 after intratracheal injection, QFHXD-413.3 mg/kg, -826.6 mg/kg, and -1239.9 mg/ kg groups received intragastric administration of 413.3, 826.6, and 1239.9 mg/kg of QFHXD, respectively, and the prednisone group received 0.25 mg/kg of prednisone daily for 28 consecutive days. Intragastrical injections of normal saline were administered to both the control and fibrosis groups. For further research, all rats were slaughtered on day 28 and their lung tissues were collected.

Hematoxylin and eosin (H&E) and masson's trichrome (MT) staining
Following fixation with 4% paraformaldehyde for 24 hr, dehydration, and embedding in paraffin, sections of the lungs (5-µm thickness) were processed for H&E and MT staining. The stained lung tissue sections were observed under an upright fluorescence microscope (200 × magnification, Olympus, Tokyo, Japan). The Alveolitis score was used to semi-quantitatively assess histological alterations in lung tissues as a result of inflammation of the alveoli (18). Alveolitis was assessed using H&E-stained sections based on the following criteria: none (0): absence of alveolitis; mild (1+): an infiltrate of mononuclear cells thickening the alveolar septum; involvement confined to focal, basal pleural lesions covering ≤ 20 % of the lung area, well preserved alveolar architecture; moderate (2+): Alveolitis that covers 20-50% of the lung, with a pleural emphasis; severe (3+): Diffuse alveolitis affecting half of the lungs, with occasional solid mononuclear cells in the alveoli, and interstitial and/or alveolar bleeding. Based on the ratio of pulmonary collagen-positive in MT staining (blue) in histological sections, Image J software (National Institutes of Health, MD, USA) was used to quantify PF.

Statistical analysis
The values were mean + standard deviation (SD). Oneway ANOVA was conducted for statistical analysis using Prism 9.0 (GraphPad Software, CA, USA). The results were deemed statistically different when P<0.05.

QFHXD ameliorated alveolus inflammation and BLMinduced PF
As shown in Figure 1A, lung tissues exhibited histological changes. Within the control group, there was an integral lung structure and normal alveolar septum, whereas the fibrosis group indicated thickened and widened alveolar septum, collapsed alveolus fusion, and a significant number of inflammatory cell infiltrations. Furthermore, in the fibrosis group, alveolitis was considerably more severe compared with the control group (P<0.01). Clearly, QFHXD administration improved alveolitis, particularly in the QFHXD-1239.9 mg/kg group (vs fibrosis group, P<0.05).
Similarly, in the control group, MT staining revealed a few blue collagen fibers in the lung tissues. In contrast, in the fibrosis group, there were many blue collagen fibers, and the collagen ratio was significantly higher (P<0.01). QFHXD administration considerably decreased the collagen ratio (QFHXD-826.6 mg/kg and QFHXD-1239.9 mg/kg vs fibrosis group, P<0.05 and P<0.01, respectively, Figure 1B).

QFHXD suppressed the expression of collagens and marker proteins
The protein expressions of representative collagen, such as collagen I, and collagen III (Figure 2A), were analyzed to determine the effect of QFHXD on collagen deposition. In contrast with the control group, collagen I expression was  Figure 2B), while QFHXD (particularly in QFHXD-826.6 mg/kg and QFHXD-1239.9 mg/kg groups) and prednisone administration down-regulated the protein expression of collagen I in PF rats (P<0.01). Similarly, the collagen III protein expression was higher in the fibrosis group than in the control group (P<0.01), while all treatment groups decreased the collagen III protein expression dramatically (vs fibrosis group, P<0.01, Figure 2C).
The expressions of vimentin and α-SMA, which indicate the progression of PF (19), are in Figure 3A. In BLM-induced rats, BLM treatment markedly increased the expression of vimentin and α-SMA (vs control group, P<0.01), whereas QFHXD and prednisone treatment decreased the expression of these proteins (P<0.01, Figure 3B and 3C).

QFHXD down-regulated ACE-AngII-AT1R and upregulated ACE2-Ang-(1-7)-Mas axis
AGT is the only substrate for all Ang peptides. It has been reported that AGT is up-regulated in the AEC of BLM-induced rats (20). Herein, the protein expressions of ACE, AGT, and AT1R were shown in Figure 4A. BLM induction evidently increased the levels of ACE, AGT, and AT1R proteins (vs control group, P<0.01). Nevertheless, QFHXD and prednisone decreased ACE ( Figure 4B), AGT ( Figure 4C), and AT1R ( Figure 4D) protein expressions (vs fibrosis group, P<0.01). Between QFHXD-413.3 mg/kg and the fibrosis group, there was no difference in AT1R protein expression.
As can be seen in Figure 5A, ACE2 and Mas were expressed. Instead, the expression of ACE2 protein was down-regulated in the fibrosis group (vs control group,     Figure 5B). Similarly, Mas protein level was also obviously down-regulated in the BLM-induced fibrosis group (vs control group, P<0.01). However, QFHXD and prednisone intervention considerably up-regulated the Mas expression in the BLM-induced rats (P<0.01, Figure 5C).

QFHXD reduced the overexpression of NOX4 and promoted the expression of Nrf2
The protein expressions of NOX4 and Nrf2 were indicated in Figure 6A. As a result of BLM stimulation, there was a dramatic decrease in Nrf2 protein value in the fibrosis lung tissues (vs control group, P<0.01), while the QFHXD-1239.9 mg/kg group markedly up-regulated the Nrf2 protein expression (vs fibrosis group, P<0.05, Figure 6B). In contrast, BLM induction up-regulated the expression of NOX4 protein in the control tissues (P<0.01). Apart from the QFHXD-413.3 mg/kg group, all treatment groups showed significant down-regulation of NOX4 protein overexpression (P<0.05, Figure 6C), particularly in the QFHXD-1239.9 mg/kg group (P<0.01).
The potential mechanism of QFHXD exerting on PF was shown in Figure 7. As a result of AEC apoptosis and alveolar fibrosis, PF could be induced by BLM. QFHXD may attenuate collagen deposition and marker protein expressions to further protect PF by suppressing oxidative stress by balancing the RAS system.

Discussion
There are obvious advantages of Traditional Chinese Medicine (TCM) in the process of treating PF, due to its rich experience (21). A previous study has shown that the Jinshui Huanxian formula (JHF) could suppress oxidative stress by restoring the balance of Nrf2-NOX4 in the treatment of PF (22). QFHXD, a Chinese medicine formula constructed based on the characteristics of PF patients has been proven effective in alleviating clinical symptoms and relieving inflammatory response and fibrosis deposition. In this study, BLM was used to establish the PF rat models. QFHXD was administered to BLM-induced PF rats at 413.3, 826.6, and 1239.9 mg/kg doses to determine its therapeutic effect. This study found that QFHXD significantly down-regulated the ACE-AngII-AT1R axis and up-regulated the ACE2-Ang-(1-7)-Mas axis, achieving the RAS system balance, subsequently exhibiting anti-oxidative stress property via lowering NOX4 level and facilitating Nrf2 growth in lung tissues. Additionally, QFHXD suppressed alveolus inflammation, collagen protein expressions, vimentin, and α-SMA expressions to inhibit PF.
PF is a chronic lung disease characterized by inflammation of lung tissues. When collagen synthesis and degradation are imbalanced, fibrosis is characterized by an increase in collagen fragments, which may have pro-inflammatory effects (23,24). Therefore, PF can be ameliorated by inhibiting alveolar inflammation and inflammatory responses. Using H&E staining, we observed alveolar structure disruption and inflammatory cell infiltration in the fibrosis tissues. The alveolitis was ameliorated after administration with a 1239.9 mg/kg dose of QFHXD. Additionally, QFHXD significantly attenuated the fibrosis deposition in groups receiving 826.6 and 1239.9 mg/kg doses of QFHXD with MT staining. It can be speculated that QFHXD could reduce alveolar inflammation and fibrosis deposition, thus exerting a therapeutic effect on PF.
Alveolar epithelium injury and abnormal repair are critical for PF initiation (25). ECM, which consists of fibrillar collagen and fibronectin, promotes alveolar epithelial type II cell (AT2) regeneration failure in lung injury repair (26). The major ECM proteins (collagen and elastin) serve as scaffolding for cells and tissues. During wound healing and fibrosis, collagen I is synthesized initially and at a quicker rate than collagen III. As a consequence, the composition of the ECM can affect the stiffness of lung tissues due to the different ratios of collagen I to III (27,28). In addition, large quantities of ECM proteins, such as collagen I and extra domain A (EDA) fibronectin, are secreted by α-SMA, whose deposition is crucial in the process of wound repair (29). A previous study has suggested that vimentin could coordinate important cellular activities that control wound healing, such as collagen accumulation (30). Herein, a QFHXD treatment decreased the expression of ECM  proteins and fibrosis-linked proteins in lung tissues of rats induced by BLM. QFHXD administration attenuated these protein expressions in a dose-dependent manner. These results suggested that QFHXD may have an anti-fibrotic effect partly by attenuating ECM deposition.
Additionally, AT2 injury/dysfunction and even death can lead to loss of lung function and contribute to IPF (31,32). The RAS system is made up of ACE-AngII-AT1R and ACE2-Ang-(1-7)-Mas axes. Important consequences for early PF development and progression are discovered to be associated with imbalances in these two axes. During BLM-induced PF, elevated levels of ACE, AngII, and AT1R were strongly associated with disease progression (33). Nevertheless, overexpression of ACE2 and Ang-(1-7) may protect against BLM-or AngII-induced PF by suppressing the MAPK/NF-κB pathway (12). In this study, QFHXD up-regulated ACE2 and Mas expression levels, while correspondingly weakening the expression levels of ACE, AGT, and AT1R. Intratracheal injection of AGT antisense oligonucleotide (ASO) has been shown to inhibit AGT synthesis, AEC death, and collagen accumulation in BLMinduced PF Wistar rats (34). The results of this study revealed that QFHXD balanced the RAS system by switching the ACE-AngII-AT1R to the ACE2-Ang-(1-7)-Mas axis, which helped stop the progression of PF.
BLM-induced PF appears to result from an inflammatory lesion accompanied by a macrophage and neutrophil accumulation in the lower respiratory tract (35). In this lesion, activated inflammatory cells may accumulate and release harmful amounts of ROS. This may be involved in parenchymal injury and alveolar fibrosis (36). Both overexpression of NOX4 and low expression of Nrf2 lead to impaired redox homeostasis, which contributes to oxidative stress associated with PF metabolism (37). NOX4 plays a crucial role in regulating intracellular ROS production. Thus, inhibition of NOX4 activity to regulate intracellular ROS generation has become a major therapeutic approach to treating fibrosis (38). Moreover, in the BLM-induced PF model, Nrf2 regulates anti-oxidant production and defense enzyme expression, thereby protecting PF against oxidative damage (39). In the present study, QFHXD administration achieved Nrf2-NOX4 redox homeostasis by down-regulating NOX4 protein expression and upregulating the Nrf2 protein level, which had a protective effect against lung injury and PF. An earlier study has demonstrated that alveolar injury, α-SMA up-regulation, and ECM component secretion and deposition were NOX4-dependent in a BLM-induced PF model and that NOX4 inhibitors markedly reduced the established fibrotic response (40). Nrf2 expression is negatively correlated with α-SMA and collagen I expression, and thus activating Nrf2 promotes anti-oxidant defense against IPF (41). According to the findings in this study, QFHXD may reduce alveolar injury and collagen deposition, as well as marker protein expressions, by inhibiting NOX4 expression and facilitating Nrf2 expression.
Additionally, AngII-induced NOX-dependent superoxide activation is a major pathogenic factor in PF progression (42). Yue et al. found that hyperoxic lung injury oxidative stress was attenuated through ACE2 regulation of the Nrf2 pathway as well as its downstream anti-oxidant enzymes (43). These observations first revealed that QFHXD exerted anti-oxidant effects by up-regulating the ACE2-Ang-(1-7)-Mas axis and down-regulating AngII expression, as well as the ACE-AngII-AT1R axis to delay PF progression. Thus, it was speculated that QFHXD may further protect PF by inhibiting oxidative stress by balancing the RAS system. Nevertheless, it is still unclear how QFHXD inhibits oxidative stress by balancing these two axes and thus reduces fibrosis. Therefore, the underlying mechanism needs to be further investigated.
There are some limitations to this study. As QFHXD was superior to prednisone in anti-fibrosis studies for PF animal models, further studies will be necessary to speculate on the influence of QFHXD in treating PF patients clinically due to the difference between animal models and human PF patients. This study was limited to the lack of inflammatory factors and further signaling pathway studies.

Conclusion
This study revealed that as a constructed Chinese medicine formula, QFHXD, was effective at suppressing inflammation-induced infiltration of lung tissues and PF by reducing oxidative stress through restoring the balance of ACE-AngII-AT1R and ACE2-Ang-(1-7)-Mas axes. The findings suggested that QFHXD might be a viable clinical therapy for PF.